Abstract: Excessive alcohol use damages many organs, but most commonly results in alcoholic liver disease (ALD). The molecular mechanisms by which ethanol (EtOH) exerts its damaging effects are extensively studied, but not fully understood, thus hampering the development of therapeutic options for the treatment of ALD. Furthermore, to date, there are no experimental animal models that recapitulate the full spectrum of ALD symptoms. The pregnane X receptor (PXR), expressed primarily in the liver and intestine, is a xenobiotic nuclear receptor that defends against toxic agents. PXR activation in humans and rodents leads to hepatic triglyceride (TG) accumulation, one feature of ALD, suggesting that the PXR may be involved in ALD pathogenesis. However, the role of mouse and human PXRs in EtOH hepatotoxicity has not been characterized. The earliest sign of liver injury observed in alcoholics is steatosis. While alcoholic steatosis progresses to alcoholic steatohepatitis during the development of ALD, the mechanisms that contribute to this deterioration are unclear. However, we have intriguing preliminary data suggesting the possibility that PXR is involved in the progression of alcoholic steatosis to more severe liver disease. Basal hepatic TG levels were significantly higher in male mice that lacked the PXR (PXR-KO) than in wild type (WT) or PXR-humanized (hPXR) transgenic mice; conversely WT and hPXR mice accumulated higher levels of TG when treated with EtOH and EtOH-treated hPXR mice were most prone to liver injury. The objectives of this R01 proposal are: 1) to explore the underlying molecular mechanisms by which PXR signaling regulates EtOH-induced TG accumulation and related lipid metabolic pathways and 2) to identify the molecular features of hPXR vs. WT mice that contribute to their increased susceptibility to EtOH-induced liver injury. Based upon our compelling preliminary data, our central hypothesis is that the nuclear receptor PXR is required for both ethanol-induced steatosis and progressive hepatotoxicity. We will carry out molecular and cellular studies using the three mouse models (WT, PXR-KO, and hPXR) and hepatocytes isolated from these animals to achieve the following aims. Specific Aim 1 will monitor i) the effect of PXR signaling on lipid synthesis, oxidation and export in hepatic steatosis and ii) corresponding changes in lipid composition in plasma and liver after ethanol ingestion. Specific Aim 2 will determine whether PXR regulates the main enzyme that metabolizes EtOH, ADH gene transcription and how it affects ethanol and acetaldehyde elimination rates. Specific Aim 3 will investigate the role of PXR signaling in hepatocyte proliferation following acute and chronic EtOH exposure. Our proposed study is novel in its use of humanized mice to examine the function of PXR in ALD, providing data that can be more directly translated into clinical research. This study is also significant in a) providing valuable insights into the role of mouse and human PXRs in ethanol hepatotoxicity via their regulation of alcohol metabolism and liver regeneration, and b) offering new therapeutic targets for the treatment of ALD.